Although originally considered mysterious and 'sphinx-like', glycosphingolipids (GSLs) and related sphingolipid (SL) metabolites have emerged as key bioactive signaling molecules and organizers of membrane micro- domains, playing vital roles in normal (cell development, differentiation, apoptosis, autophagy) and pathogenic membrane-related processes (bacterial &viral infections, oncogenesis, genetic lipid storage diseases). However, critical issues persist regarding exactly how lipid compositional changes involving both SLs and non- SL lipids: i) alter the membrane physical environment;ii) control SL lateral organization and surface accessi- bility;iii) regulate the translocation and functionality of peripheral, amphitropic membrane proteins with affinity for SLs. Our goal is to address these key issues by defining the functional regulation of a novel lipid transfer protein family that selectively transfers SLs between membranes using a unique lipid-binding conformational fold (GLTP-fold) and a new membrane interaction motif for peripheral, amphitropic proteins. The objective of this renewal application, which builds on our progress and exciting new Prelim. Data, is to move the field forward by accomplishing the following Aims: 1) Map Key Regions of GLTP-folds that Control Selectivity for Different Types of Sphingolipids;2) Characterize Novel Membrane Interaction Motif of GLTP-folds;3) Determine How Lipid-Sphingolipid Lateral Packing Interactions within Membranes Regulate GLTP-fold Action;4) Determine How Human GLTP Up-Regulation Induces Cell Death Processes. We are particularly well prepared to undertake the proposed research because we achieved the first successful cloning of GLTP as well as the first three-dimensional structure of GLTP, establishing the GLTP-fold as unique among lipid binding/transfer proteins, as the prototype for the new GLTP superfamily, and as having a novel membrane interaction domain among peripheral amphitropic proteins. We also have developed the means to quantita- tively assess lateral elastic interactions of GSLs with each other and with other membrane lipids while achieving nanoscale resolution into lipid lateral mixing, enabling fundamental insights into the structural features of sphingolipids that control interaction with GLTP. Because of our progress, the complementary expertise within our research team, and the historic success of our collaborators, the total research environment is especially conducive for achieving the new aims proposed herein. It is our expectation that our new discovery of a human GLTP-fold that binds/transfers ceramide-1-phosphate will not only strongly under- score the importance of GLTP-folds in human cells, but that the new knowledge will have the added significant outcome of enabling production of 'designer GLTP-folds'with highly focused selectivity for specific glycolipid/sphingolipid types by point mutation. This new knowledge is expected to provide a foundation for biotechnological use of the GLTP-fold in exciting and innovative ways, such as introducing specific GSL antigens into cells to help achieve targeted destruction of diseased cells via immunotherapeutic means. PUBLIC HEALTH RELEVANCE: The new knowledge gained by the research proposed herein is expected to provide the foundation for using glycolipid transfer proteins in new and innovative ways, such as introducing specific GSL antigens into cancer cells to help achieve targeted destruction of diseased cells via immunotherapeutic as well as for triggering self- destruction of diseased cells by programmed cell death processes.